Aminoglycoside ntibiotics are one of the most commonly prescribed treatments for serious infections, and continue to remain popular due to their effectiveness against multi-drug resistant bacteria. Aminoglycosides preferentially bind to the near-universally conserved decoding site of the bacterial ribosome, where proper matching of the mRNA codon and tRNA anticodon occurs. Key distinctions in the decoding site architecture between bacterial and human ribosomes determine aminoglycoside specificity. Point mutations in the human mitochondrial ribosome reduce the number of specificity determinants. Correspondingly, such mutations are associated with aminoglycoside-induced side effects in humans, commonly resulting in permanent hearing loss. While significant progress has been made towards understanding how aminoglycosides recognize the bacterial ribosome decoding site: 1) the molecular mechanism of translation, 2) the specific steps of the process that are targeted by aminoglycosides and 3) how alterations in this region of the ribosome confer specificity while retaining ribosomal functions remain obscure. Here, using single-molecule Fluorescence Resonance Energy Transfer (smFRET) imaging methods, quantitative biophysical investigations of the translation mechanism are proposed that will enable unprecedented insights into the molecular mechanism of translation in both aminoglycoside -sensitive and -resistant ribosomes. These efforts will ultimately lead to development of a platform that can potentially be used to develop new strategies to mitigate unwanted side effects of these important antibiotics. Towards this goal, methods that have been recently employed to describe a complete kinetic mechanism of the bacterial translation elongation cycle will be applied to bacterial ribosomes engineered to contain human wild type and deafness mutant decoding sites. Such efforts will lead to a significantly deeper understanding of how these structural distinctions alter the translation mechanism and enable the molecular origins of aminoglycoside hypersensitivity to be delineated. As the proposed investigations require orders of magnitude less material than traditional biophysical methods, analogous investigations will also be performed on isolated human mitochondrial ribosomes that build upon this foundation. The successful completion of the research AIMs will yield significant insights into the role of mutations in aminoglycoside-induced ototoxicity and the mechanism of mitochondrial translation, as well as a much-needed platform that can potentially be used to develop safer aminoglycoside antibiotics. PUBLIC HEALTH RELEVANCE: The proposed research aims to significantly advance our understanding of the origins of antibiotic specificity for the bacterial ribosome and provide insights into the molecular mechanism of aminoglycoside hypersensitivity associated with mutations in the human mitochondrial ribosome, which often result in permanent hearing loss. Through the implementation of state-of-the-art single-molecule imaging techniques, the present study will provide novel platforms by which to directly investigate the performance of mitochondrial translation that can ultimately be used to design tailored aminoglycoside antibiotics with fewer side effects.